Sterile Sand Research Articles

Host Range and Symbiotic Effectiveness of N2O Reducing Bradyrhizobium Strains.

Emissions of the potent greenhouse gas N2O is one of the environmental problems associated with intensive use of synthetic N fertilizers, and novel N2O mitigation strategies are needed to minimize fertilizer applications and N2O release without affecting agricultural efficiencies. Increased incorporation of legume crops in agricultural practices offers a sustainable alternative. Legumes, in their symbiosis with nitrogen fixing bacteria, rhizobia, reduce the need for fertilizers and also respond to the need for increased production of plant-based proteins. Not all combinations of rhizobia and legumes result in efficient nitrogen fixation, and legume crops therefore often need to be inoculated with compatible rhizobial strains. Recent research has demonstrated that some rhizobia are also very efficient N2O reducers. Several nutritionally and economically important legumes form root nodules in symbiosis with bacteria belonging to Bradyrhizobium. Here, the host-ranges of fourteen N2O reducing Bradyrhizobium strains were tested on six legume hosts; cowpea, groundnut, mung bean, haricot bean, soybean, and alfalfa. The plants were grown for 35 days in pots in sterile sand supplemented with N-free nutrient solution. Cowpea was the most promiscuous host nodulated by all test strains, followed by groundnut (11 strains) and mungbean (4 strains). Three test strains were able to nodulate all these three legumes, while none nodulated the other three hosts. For cowpea, five strains increased the shoot dry weight and ten strains the shoot nitrogen content (pairwise comparison; p < 0.05). For groundnut the corresponding results were three and nine strains. The symbiotic effectiveness for the different strains ranged from 45 to 98% in cowpea and 34 to 95% in groundnut, relative to fertilized controls. The N2O reduction capacity of detached nodules from cowpea plants inoculated with one of these strains confirmed active N2O reduction inside the nodules. When released from senescent nodules such strains are expected to also act as sinks for N2O produced by denitrifying organisms in the soil microbial community. Our strategy to search among known N2O-reducing Bradyrhizobium strains for their N2-fixation effectiveness successfully identified several strains which can potentially be used for the production of legume inoculants with the dual capacities of efficacious N2-fixation and N2O reduction.

Interactions between putatively endophytic bacteria and tall fescue (Festuca arundinacea): plant growth promotion and colonization in host and non-host cultivars

Tall fescue (Festuca arundinacea) is a forage grass highly used in prairies. Cultivar SFRO Don Tomas has excellent drought and cold resistance, characteristics which are influenced by the native endophytic bacterial community. The aims of this work were to deepen the characterization of a selected group of putatively endophytic strains with different plant growth promoting (PGP) traits and to analyze the relationship between colonization and PGP abilities. PGP with single inoculations in pots containing a mixture of sterile sand and field soil and with paired inoculations in field soil cores were carried out. Colonization of plants inoculated singly or co-inoculated under gnotobiotic conditions was quantified. Confocal laser microscopy was used to visualize the colonization and infection processes of strain Streptomyces sp. UYFA156 in cultivars SFRO Don Tomas and Tacuabe. Tall fescue cultivars displayed different responses to single bacterial inoculation. Inoculation with two strain pairs which included UYFA156 were successful in promoting growth in soil cores. Higher bacterial densities did not correspond to higher PGP. Strain UYFA156 was observed colonizing both fescue cultivars. UYFA156 is a true endophyte of tall fescue. Its PGP activity is cultivar-specific and may be related to the modulation of other endophytes.

Khelatisasi Ion Aluminium oleh Asam Organik Eksudat Akar Brachiaria

Aluminum toxicity is one of the major factors inhibiting plant growth in acid soils. Brachiaria grass adapt to high Al concentration. This experiment was conducted to study exudation of low molecular weight organic acids (LMWOA) activated by Al, from Brachiaria roots and its potential in chelating Al. Three Brachiaria species, i.e. B. decumbens, B. ruziziensis and B. brizantha, planted in sterile sand culture and were treated with 5 Al concentrations (0, 100, 200, 300 and 400 μM). After two-month experiment, three kinds of LMWOA, i.e, malic, citric, and oxalic acids, produced by the three Brachiaria-root exudates were measured in the sand culture. The production of malic acid was higher than that of citric and oxalic acid. Those organic acids were influenced by Al concentration; the higher Al concentration the higher organic acid content would be. The organic acids were also proved to form Al-organic compounds effectively of which B. decumbens and B. brizantha were more effective in chelating Al at relatively low Al (100 μM) and at relatively high Al concentration (300 μM and 400 μM), respectively.

Physical constraints for respiration in microbial hotspots in soil and their importance for denitrification

Abstract. Soil denitrification is the most important terrestrial process returning reactive nitrogen to the atmosphere, but remains poorly understood. In upland soils, denitrification occurs in hotspots of enhanced microbial activity, even under well-aerated conditions, and causes harmful emissions of nitric (NO) and nitrous oxide (N2O). The timing and magnitude of such emissions are difficult to predict due to the delicate balance of oxygen (O2) consumption and diffusion in soil. To study how spatial distribution of hotspots affects O2 exchange and denitrification, we embedded microbial hotspots composed of porous glass beads saturated with growing cultures of either Agrobacterium tumefaciens (a denitrifier lacking N2O reductase) or Paracoccus denitrificans (a “complete” denitrifier) in different architectures (random vs. layered) in sterile sand that was adjusted to different water saturations (30 %, 60 %, 90 %). Gas kinetics (O2, CO2, NO, N2O and N2) were measured at high temporal resolution in batch mode. Air connectivity, air distance and air tortuosity were determined by X-ray tomography after the experiment. The hotspot architecture exerted strong control on microbial growth and timing of denitrification at low and intermediate saturations, because the separation distance between the microbial hotspots governed local oxygen supply. Electron flow diverted to denitrification in anoxic hotspot centers was low (2 %–7 %) but increased markedly (17 %–27 %) at high water saturation. X-ray analysis revealed that the air phase around most of the hotspots remained connected to the headspace even at 90 % saturation, suggesting that the threshold response of denitrification to soil moisture could be ascribed to increasing tortuosity of air-filled pores and the distance from the saturated hotspots to these air-filled pores. Our findings suggest that denitrification and its gaseous product stoichiometry depend not only on the amount of microbial hotspots in aerated soil, but also on their spatial distribution. We demonstrate that combining measurements of microbial activity with quantitative analysis of diffusion lengths using X-ray tomography provides unprecedented insights into physical constraints regulating soil microbial respiration in general and denitrification in particular. This paves the way to using observable soil structural attributes to predict denitrification and to parameterize models. Further experiments with natural soil structure, carbon substrates and microbial communities are required to devise and parametrize denitrification models explicit for microbial hotspots.

PENGARUH PEMUPUKAN DAN KEDALAMAN GAMBUT PADA PRODUKSI INOKULUM MIKORIZA ARBUSKULA DALAM PENANGKARAN DENGAN INANG Sorghum bicolor (L.) Moench

The research was to obtain information about the combination of fertilizers in the production of arbuscular mycorrhizal inoculums of various depths. This research is using an experimental method. The research uses divided plot design (RPT) with a basic RAK pattern. The treatment factor consists of the main plot (peat depth 0-50 cm and 50-100 cm) and subplot is a combination of fertilizer (P1 = without fertilizing; P2 = NPK+rubberwood vinegar; P3 = NPK+peat vinegar; P4 = NPK+fern-root vinegar). Spore breeding process is ± 3 months. Hosts use sorghum with sterile sand media. The observation variable consisted of the percentage of root infections and the number of spores. Data analysis using ANOVA. Identification of spores based on morphology and Melzer reaction. The results showed that fertilization factors affected the number of spores. The number of spores without fertilization was higher, namely 1642,500 spores compared to combination fertilizers (NPK+fern-root vinegar was 707,667 spores; NPK+rubberwood vinegar was 606,833 spores; NPK+peat vinegar was 206,667 spores). The rate of 0-50 cm colonization without fertilization, NPK+peat vinegar, NPK+rubberwood vinegar is included in the high category. Then in the treatment at 50-100 cm without fertilizing, NPK+peat vinegar, NPK+fern-root vinegar infection rates are also high. The type of spore found is Glomus sp.1; Glomus sp.2; Glomus sp.3; Glomus sp.4. The results of the number of spores and the percentage of root infections showed that organic vinegar was known to not stimulate FMA sporulation, but organic vinegar did not inhibit FMA colonization.Keyword: Mycorrhizal, organic vinegar, peat depth, sorghum

Respuesta de Zea mays a Burkholderia spp endófita de Zea mays var mexicana (teocintle)

Zea mays var mexicana or teocinte is a grass, considered one of the main ancestors of Zea mays. Teocinte, like other grasses, is associated with genera of endophytic plant growth promoters (EPGPB) in the interior of plant tissues. Z. mays is a domestic grass normally cultivated under the intensive production system, with relatively high doses of nitrogen fertilizer (NIFE), a possible alternative solution to minimize this problem, is the inoculation of the seed of Z. mays with genera of EPGPB teocinte, which colonize the roots, stems, leaves and promote a healthy growth at a reduced dose up to 50% NIFE such as urea, which is equivalent to decrease from 280 kg/ha-1 to 140 kg/ha-1, of what is recommended for the region of northern Michoacan and southern Guanajuato, Mexico. The objectives of this work were: i) to determine the density of Burkholderia spp BEPCV of teocinte, ii) to analyze the effect of the inoculation of Burkholderia spp EPGPB on the growth of Z. mays, at reduced dose of NIFE in greenhouse, iii) identify Burkholderia spp BEPCV teocinte. The population density of Burkholderia spp EPGPB was determined in tissues (organs), seeds, stem-leaves and roots of teocinte by sowing them on Pseudomonas cepacia azelaic acid tryptamine (PCAT), after incubation. Burkholderia spp colonies were selected for abundance in: leaves, stem, roots and/or seeds. These isolates of Burkholderia spp, were coded with the keys T41 and T42 and inoculated in Z. mays sown in sterile sand, where as the only variable-response the total dry weight was determined compared to the Z. mays cultivated with the recommended NIFE dose, for that region, it was concluded with the biochemical characterization. The results show that in Burkholderia spp BEPCV of teocinte, a density of 3.4 Log CFU/g of fresh weight (GPF) was registered in the root, of 3.0 Log CFU/GPF in the seed and a similar value in the stem-leaves of teocinte The response of Z. mays to Burkholderia T41 caused an increase of 47% in the dry weight of the root to the tillering, and of 57% to the extension of the stem, compared with the Z. mays without inoculating with the maximum level of NIFE (urea) 280 kg/ha-1. The biochemical identification profile of Burkholderia spp supports a genetic relationship with B. vietnamiensis. It is concluded that teocinte is a source of EPGPB for sustainable production Z. mays at a reduced dose of NIFE.

Potency of nitrogen fixing bacteria isolated from POME disposal pond and their effect on the growth of Caesalpinia pulcherrima (L) Sw

Nitrogen fixing bacteria (NFB) play an important role to promote plant growth through nitrogen fixation and IAA hormone production. The objective of this research was to obtain the functional bacteria (nitrogen fixing bacteria) isolated from POME disposal pond that show the ability in nitrogen fixation and the potential as plant growth promoting rhizobacteria (PGPR), as well as to investigate the potential of these bacteria through in vitro and in vivo growth of Caesalpinia pulcherrima (L) Sw, and its effect on bacterial populations in media contaminated by POME. In vitro plant growth promoting activity characterization included N-fixing activity, Indole Acetic Acid production, and C. pulcherrima germination (inoculant + POME). In vivo plant growth promoting activity used sterile sand inoculated with NFB and watered with POME, whereas the control was not inoculated and watered with aquadest. Five strains were found as potential PGPR, namely Azotobacter sp., Azospirillum sp1-3., and Rhizobium sp. In addition, Azospirillum sp.1 is the best PGPR that produced highest seed vigour index (SVI), seed germination in vitro, and seed germination in vivo respectively. Azospirillum sp. 1 and Rhizobium sp. have the ability to promote growth of C. pulcherrima in vitro and in vivo with bacterial populations as much as 107cfu mL−1 in POME contaminated media. The bacteria and C. pulcherrima are potential for reclaiming infertile soil.

First Report of Sclerotinia Crown Rot Caused by Sclerotinia minor on Hemp

HomePlant DiseaseVol. 103, No. 7First Report of Sclerotinia Crown Rot Caused by Sclerotinia minor on Hemp PreviousNext DISEASE NOTES OPENOpen Access licenseFirst Report of Sclerotinia Crown Rot Caused by Sclerotinia minor on HempS. T. Koike, H. Stanghellini, S. J. Mauzey, and A. BurkhardtS. T. Koike†Corresponding author: S. T. Koike; E-mail Address: SKoike@trical.comhttp://orcid.org/0000-0002-3184-3778TriCal Diagnostics, Gilroy, CA 95020Search for more papers by this author, H. StanghelliniTriCal Diagnostics, Gilroy, CA 95020Search for more papers by this author, S. J. MauzeyTriCal Diagnostics, Gilroy, CA 95020Search for more papers by this author, and A. BurkhardtTriCal Diagnostics, Gilroy, CA 95020Search for more papers by this authorAffiliationsAuthors and Affiliations S. T. Koike † H. Stanghellini S. J. Mauzey A. Burkhardt TriCal Diagnostics, Gilroy, CA 95020 Published Online:16 May 2019https://doi.org/10.1094/PDIS-01-19-0088-PDNAboutSectionsSupplemental ToolsAdd to favoritesDownload CitationsTrack Citations ShareShare onFacebookTwitterLinked InRedditEmailWechat In summer 2018, commercial field grown hemp (Cannabis sativa L.) plants in San Benito County, CA, showed signs of a disease. Leaves initially wilted and turned dark green. Foliage eventually dried up completely. The base of the plant crown in contact with soil supported the growth of white to gray mycelium and small (0.5 to 3 mm diameter), irregularly shaped, black sclerotia. Crown tissue beneath the epidermis was necrotic. However, plant roots appeared symptomless. To estimate disease incidence, eight replicates of 100 plants each were assessed for collapse symptoms and sclerotia on plant crowns. Overall disease incidence was limited and was approximately 1%. To test for pathogens, symptomatic crown pieces were surface sterilized in 0.006% NaOCl for 2 min and plated on acidified (2 ml of 25% lactic acid/liter) corn meal agar. After 2 to 3 days, rapidly growing, colorless, appressed mycelium emerged from crown pieces. Mycelial transfers to potato dextrose agar (PDA) plates resulted in white to gray colonies that produced profuse numbers of black sclerotia typical of Sclerotinia minor (Koike et al. 2007). Sclerotia that were removed from the collected field plants, dipped in ethanol, rinsed, and plated on acidified PDA resulted in colonies that again appeared typical of S. minor. To test for pathogenicity, four isolates were grown on PDA until sclerotia were fully developed and surface mycelium was dry. For each isolate, 10 sclerotia were removed from the PDA plates and placed adjacent to the crowns of eight 2-month-old potted hemp plants approximately 1 cm below the soil surface. The sclerotia were then covered lightly with potting medium. Plants were maintained under ambient light and a daytime temperature range of 19 to 24°C. After 2 weeks, leaves of inoculated plants began to wilt and crown tissue was necrotic. Isolation from symptomatic crowns resulted in colonies that morphologically and molecularly were identical to the original isolates. Control plants, inoculated with sterile sand, developed no foliar or crown symptoms. For species identification, DNA was extracted using the DNAeasy Plant kit (Qiagen) from mycelium scraped from the surfaces of 21-day-old cultures of both the original and postinoculation isolates. DNA was amplified and sequenced using the ITS1 and ITS4 primers for the internal transcribed spacer region. Using the NCBI nucleotide BLAST, original (MK356550) and postinoculation (MK356551) isolates matched S. minor (KC836493.1) with 100% query cover and 100% identity. Based on these molecular and morphological data, the isolates were identified as S. minor Jagger. This is the first report of a crown rot disease on hemp caused by S. minor, which we designate as Sclerotinia crown rot. Hemp is known to be a host of Sclerotinia sclerotiorum (Lib.) de Bary, which causes hemp canker disease on crowns and stems (McPartland 1996).The author(s) declare no conflict of interest.

Eurypsychrophilic Pseudomonas spp. isolated from Venezuelan tropical glaciers as promoters of wheat growth and biocontrol agents of plant pathogens at low temperatures

Andean tropical glaciers are disappearing rapidly and, consequently, the microbes immured in these frozen environments will be lost forever. Some of these microbes are thought to be potentially useful to develop biotechnological products or processes. Among these microbes, plant-growth promoting (PGP) bacteria have been proposed as valuable tools to develop cold-active biofertilizers and/or biopesticides. A few years ago, we hypothesized that bacteria immured within glacial ice could be effective in promoting plant growth and/or in protecting plants from pathogen infection, at low temperatures. In this study, we aimed at testing some of these traits, with a suitable plant model (Triticum aestivum). In the present study, from a collection of bacteria isolated from Venezuelan tropical glaciers, we selected four Pseudomonas isolates and tested their PGP effects at low temperatures, both in vitro and on wheat plantlets. The isolates grew well over a wide range of low temperatures and were thus classified as eurypsychrophilic. They also displayed well-known PGP traits: solubilization of inorganic phosphates, production of phytohormones and antagonism against a phytopathogenic oomycete (Pythium ultimum). Inoculation of T. aestivum seeds with some of these Pseudomonas spp. isolates promoted a significant elongation of their roots and shoots. This was also the case when wheat plantlets were grown in sterile sand or soil, at 15 °C. Inoculation of wheat seeds also protected plantlets against the damage caused by P. ultimum. Together, our results suggest that some of these Pseudomonas spp. isolates could act as cold-active biofertilizers and/or biocontrol agents.

Indirect Regeneration and Assessment of Genetic Fidelity of Acclimated Plantlets by SCoT, ISSR, and RAPD Markers in Rauwolfia tetraphylla L.: An Endangered Medicinal Plant.

Rauwolfia tetraphylla L. is an important medicinal plant species which is well known for its pharmaceutically important alkaloids. In the present study, we are reporting about its conservation by in vitro clonal multiplication through the standardized protocol of indirect regeneration by using leaf and stem based callus and assessment of genetic fidelity of acclimated plantlets by start codon targeted (SCoT), inter simple sequence repeats (ISSR), and randomly amplified polymorphic DNA (RAPD) marker based analysis. Initially friable callus was induced in maximum amounts (378.7, 323.8, and 412.8 in mg) from leaf, root, and stem explants on Murashige and Skoog (MS) media supplemented with 5.0 mg/L, 3.0 mg/L of 2,4-dichlorophenoxyacetic acid (2,4-D) and 5.0 mg/L of naphthalene acetic acid (NAA), respectively. Shoot regeneration with the maximum number of shoot buds (25 and 20) was obtained from leaf and stem calluses on MS media supplemented with TDZ (0.25 mg/L) + BAP (2 mg/L). The regenerated shoots were rooted successfully with maximum rooting percentage of 98.0 on full strength MS media amended with IAA (1.0 mg/L) and IBA (1.0 mg/L). The regenerated plantlets were hardened using 2:1 ratio of sterile garden soil and sand, followed by acclimatization in field conditions with 86% of survival. SCoT, ISSR, and RAPD primers based polymerase chain reaction (PCR) analysis was carried out to check possible genetic variations in micro propagated plants in comparison with mother plant. Among the ten SCoT (S), ISSR (R), and RAPD (OPA) primers used, S2, R10, and OPA3 has given good amplification with scorable DNA bands. The results revealed that the regenerated plants did not have any polymorphism with mother plant. Hence, the in vitro regenerated R. tetraphylla plantlets were confirmed as true-to-type.

Response of two Citrullus amarus accessions to isolates of three species of Meloidogyne and their graft compatibility with watermelon

The response of two Citrullus amarus accessions, BGV0005164 and BGV0005167, was assessed against different Meloidogyne arenaria, Meloidogyne incognita, and Meloidogyne javanica isolates in pot experiments and against M. incognita in plastic greenhouse. In the pot experiments, plants were inoculated with a second-stage juvenile per cm3 of sterile sand and maintained in a growth chamber at 25 °C for 50 days. The watermelon cv. Sugar Baby was included as a susceptible control for comparison. At the end of the experiments, the number of egg masses and eggs per plant was determined, and the reproduction index was calculated as the percentage of the number of eggs produced in the C. amarus accessions with regard to that produced in the susceptible cv. Sugar Baby. In the plastic greenhouse experiment, the ungrafted watermelon cv. Sugar Baby and watermelons grafted onto each of the C. amarus accessions and onto the watermelon rootstock cv. Robusta were cultivated from May to August 2016 in plots with nematode densities from 46 to 1392 J2 per 250 cm3 of soil at transplantation. At the end of the experiment, the galling index and the number of eggs per plant were determined, and the reproduction index was calculated. Additionally, the compatibility of the two accessions with the watermelon cv. Sugar Baby and the effect on fruit quality (weight, size, shape, firmness, pH, total soluble solids, and flesh color) were assessed under a hydroponic system in a greenhouse. The commercial rootstocks cv. Cobalt and cv. Robusta were also included. All the Meloidogyne isolates produced less egg masses and eggs per plant on the accessions than on Sugar Baby. Both accessions performed as resistant against M. arenaria, and from highly to moderately resistant to M. incognita and M. javanica in pot experiments. In the plastic greenhouse experiment, both C. amarus accessions performed as resistant to M. incognita. Both C. amarus accessions were compatible with the watermelon cv. Sugar Baby, but only the BGV0005167 accession did not influence the fruit quality. Then, the BGV0005167 accession is a promising rootstock for managing the three tropical root-knot nematode species without influencing watermelon fruit quality.

Draft Genome Analysis Offers Insights Into the Mechanism by Which Streptomyces chartreusis WZS021 Increases Drought Tolerance in Sugarcane.

Drought directly affects sugarcane production. Plant growth-promoting bacteria have gained attention as growth promoters of plants under abiotic stresses. The present study focused on genome assessment of the plant-beneficial endophyte Streptomyces chartreusis WZS021 and its vital role in sugarcane plants under drought stress. Based on in vitro plant growth-promoting trait analyses, WZS021 had multiple abilities, including tolerance to drought and production of 1-aminocyclopropane-1-carboxylic deaminase, siderophores, and indole acetic acid. We confirmed root colonization of sugarcane transplants by WZS021 by a sterile sand assay and scanning electron microscopy. Plants inoculated with strain WZS021 had a positive influence on the root parameters such as length and biomass when compared to the control plants. A comparative study of the responses of two sugarcane varieties (ROC22 and B8) to different levels of drought stress in the presence or absence of WZS021 was conducted by assessing the plant chemistry. The expression of antioxidants in sugarcane leaves varied with water stress level. WZS021 inoculation improved the contents of chlorophyll, proline, and phytohormones, revealing some potential for the mechanisms by which this strain improves drought tolerance in sugarcane plants. We identified several genes that might be involved in the plant growth- and drought tolerance-promoting effects of this strain.

Arbuscular mycorrhizal fungus on the initial growth and nutrition of Coffea arabica L. genotypes

ABSTRACT The benefits of mycorrhization occur with the growth of hyphae in colonized roots by promoting an increase of the contact surface which improves the initial growth due to a better absorption of water and nutrients. The objective was to evaluate the effect of arbuscular mycorrhizal fungus Rhizophagus clarus inoculation over the initial development and nutritional response of six genotypes of Coffea arabica L. Six genotypes of Coffea arabica L. were used (MGS Aranas, H29-1-8-5, Red Catuai IAC 144, IPR 100, Catigua MG2, Paraíso H 419-1) and with arbuscular mycorrhizal fungus - AMF: Rhizophagus clarus and without the application of the fungus. For the production of coffee seedlings, seeds were placed to germinate in plastic trays with sterile sand. After germination, 10 seedlings of each genotype were transferred to 0.120 dm3 polyethylene tubes with substrate. Then the inoculation of five seedlings of each genotype with the AMF R. Clarus was performed. When the seedlings with and without inoculation with the AMF presented six pairs of leaves they were transplanted to 13-liter pots containing soil (Dystrophic red-yellow latosol). The inoculation favored the initial growth of the coffee plants and its intensity varied according to the genotypes. The genotypes H 29-1-8-5, Red Catuai IAC 144 and Catigua MG 2 were the ones that presented higher shoot dry mass, root dry mass, total dry mass and accumulation of P, in relation to MGS aranãs, Paraiso H 419-1 and IPR 100, so they are the most promising to be inoculated with the arbuscular mycorrhizal fungus.

Screening of plant growth promoting rhizobacteria (PGPR) to promote growth of soybean

Rhizobacteria is an essential component for the maintenance of soil fertility and plant growth. The objective of the study was to characterize plant growth promoting rhizobacteria (PGPR) activity such as phosphate solubilization, nitrogen-fixing, IAA production, Acc-deaminase and cellulase activity isolated from the rhizosphere and nodules of soybean and evaluate their effectiveness in stimulating soybean seedlings growth. Greenhouse assay was conducted by completely randomized design with three replications per treatment and two controls (K1-control without inoculation of bacteria and Nitrogen, K2-control inoculated with Bradyrhizobium japonicum. Pot experiments were conducted using sterile sand and supplemented with water and Muller solution, periodically. The results showed that nine rhizobacteria isolates were efficacious as potential PGPR. Greenhouse assay results showed that the highest vegetative growth yield was obtained in soybean inoculated with SWNC1 isolate, it was characterized as Bradyrhizobium spp. which gave the total nodules of 29.67; fresh weight of nodules of 0.61 g; nodules dry weight of 0.09 g; number of flowers of 23; plants dry weight of 5.57 g and plant height of 46 cm.

Ex vitro establishment and macropropagation of cassava (Manihot esculenta ‘Valencia’) to obtain disease-free rooted plants

A common practice during cassava cultivation in Costa Rica is the use of stem cuttings coming from farmers' plantations as vegetative seeds. This system has widely spread diseases all over the country, especially the phytoplasm associated “Frog Skin” disease. At Tecnologico de Costa Rica we developed a protocol for rapid multiplication of disease-free plantlets. Terminal buds obtained from in vitro plants were grown in a MS (1962) medium under four different light treatments: 100% blue light (465 nm), 100% red light (630 nm), 50-50% red-blue light and white fluorescent light (as control), all of them with a light intensity (PPFD) of 70 μmol m-2 s-1. The best plantlets for acclimatization were obtained with blue light, which resulted in thicker stems and shorter internodes. In a second experiment, in vitro plants were transferred to the greenhouse for acclimatization, where five different substrates were evaluated: coco-coir, peat moss with perlite (75-25), mix of coco-coir with peat, sterile sand and sterile mix of soil+sand. The percentage of successfully acclimated plants was above 90% for those grown with coco-coir and peat moss, 86% for mix coco-coir+peat moss, 82% for soil+sand and 60% for sand-grown plants. Acclimatized plants were taken to a greenhouse and grown in pots with coco-coir during 8 weeks; after this time, plants became fully developed. Then cuttings of 10 cm length were obtained from the top of the plant and rooted using IBA (indolebutyric acid) in four concentrations: 0, 500, 1000 and 1500 ppm. The higher number and length of roots resulted in those cuttings treated with 1000 ppm. After three weeks, the rooted cuttings were taken successfully to the field, with a mortality of 4%.

English

The study was aimed to standardise a protocol for the in vitro mass propagation of Rhinacanthus nasutus (L) Kurz, an anticancer shrub. Leaf, node and inflorescence explants were inoculated onto the Murashige and Skoog’s (MS) medium enriched with different combinations and concentrations of growth regulators. Maximum callusing percentage was achieved in leaf explants in MS medium supplemented with 5 mg/l 2,4-D. Multiple shoots were achieved from leaf, node and inflorescence explants with maximum of 25±0.42 (5 mg/l BAP + 2.5 mg/l IAA), 11±0.87 (5 BAP + 2.5 mg/l IAA) and 8±0.56 (3 mg/l BAP + 1.5 mg/l IAA) shoots, respectively. For in vitro rhizogenesis, elongated micro shoots were aseptically transferred to the half strength MS liquid medium with maximum number of 8±0.89 roots per shoot achieved in 1 mg/ml IAA fortified MS medium. The in vitro rooted micro shoots were acclimatised under laboratory conditions for two weeks by transferring to polycups containing sterile soil, sand and vermiculite (1:1:1). After two weeks, hardened plantlets were transferred to the green house for two weeks and then finally to the garden with 95% survivability. Key words: Rhinacanthus nasutus, multiple shoots, leaf callus, node, inflorescence, rhizogenesis.

First Report of Potato Cyst Nematode Globodera pallida Infecting Potato (Solanum tuberosum) in Kenya

The potato cyst nematodes (PCN) Globodera pallida (Stone, 1973) and G. rostochiensis are key pests of potato, subject to strict quarantine regulations worldwide (EPPO 2013a). Indigenous to South America, they have spread to numerous potato-growing regions around the world. G. rostochiensis was reported from Kenya in 2015 (Mwangi et al. 2015). During a nationwide survey conducted in 2016, G. pallida was detected in Kenya at an altitude of 2,349 m above sea level in Nyandarua County (0.3150195° N, 36.48328° E). Cysts were extracted from a 200 cm³ soil sample following EPPO diagnostic protocol (EPPO 2013a), and then handpicked under a stereo microscope. The PCNs recovered showed morphometric characteristics of G. pallida and are reported here. For further studies, the Nyandarua field was resampled in February 2017 to collect additional soil samples and confirm the occurrence of G. pallida. From the collected cysts, 10 cysts were inoculated on potato (Solanum tuberosum L.) ʻShangiʼ in five pots with sterile soil and sand (1:1) and grown in a screenhouse for 3 months from May to July 2017; the multiplication rate at harvest was X― = 3.6 and PCNs were recovered from potato roots and soil. Morphometric characters showed: Granek’s ratio (n = 33) ranged from 1.53 to 4.52 µm (X― = 2.78 ± 0.78 µm), and the distance from anus to vulval basin was 34.03 to 91.45 µm (X― = 52.75 ± 13.73 µm). The stylet length of the second-stage juveniles (J2s) (n = 97) ranged from 15.87 to 25.18 µm (X― = 21.87 ± 1.43), stylet knobs displayed robust tulip/anchored shape. The lengths of the hyaline tail (HT) and the true tail (TT) ranged from 15.54 to 50.44 µm (X― = 23.94 ± 4.23) and 31.02 to 79.59 µm (X― = 50.64 ± 5.71 µm), respectively. Body length (n = 40) fluctuated from 338.41 to 468.34 µm (X― = 432.23 ± 24.95). DNA amplification was performed from 14 cysts and 25 J2s using the multiplex-PCR method adapted from Bulman and Marshall (1997) and the ITS1-5.8S-ITS2 regions (Tirchi et al. 2016). PCR cycling parameters were adjusted to a 5-min initial denaturation phase and 37 PCR cycles for multiplex-PCR (EPPO 2013b). The species-specific primers ITS5/PITSp4 for G. pallida (265 bp) and AB28/TW81 primers (1,188 bp) were used to amplify the small subunit of the 18s ribosomal RNA and the ITS region, respectively; PCR-amplicons were purified using the QIAquick PCR Purification Kit (Qiagen, U.S.A.) and the DNA sequences were manually edited using BioEdit Sequence Alignment Editor; in silico analyses were conducted with the NCBI-BLAST tool. The Kenyan ITS5/PITSp4 sequences (NCBI accession no. MG309873) presented 100% similarity to the G. pallida isolates KJ409623.1 and AF016869 (score = 481; E value = 5.02e⁻¹³²), while the Kenyan AB28/TW81 sequence (NCBI accession no. MG309920) showed 95 and 94% similarity to the G. pallida isolates HF583248.1 and HQ670272.1 (score = 1,218 and 1,221; E value = 0), respectively. This first report of G. pallida in sub-Saharan Africa has paramount phytosanitary and regulatory implications for potato growers and traders, national extension services, and policy makers in Kenya and the surrounding region.

A simple and inexpensive physical lysis method for DNA and RNA extraction from freshwater microalgae

In this work, a simple and inexpensive physical lysis method using a cordless drill fitted with a plastic pellet pestle and 150mg of sterile sea sand was established for the extraction of DNA from six strains of freshwater microalgae. This lysis method was also tested for RNA extraction from two microalgal strains. Lysis duration between 15 and 120s using the cetyltrimethyl ammonium bromide (CTAB) buffer significantly increased the yield of DNA from four microalgalstrains (Monoraphidium griffithii NS16, Scenedesmus sp. NS6, Scenedesmus sp. DPBC1 and Acutodesmus sp. DPBB10) compared to control. It was also found that grinding was not required to obtain DNA from two strains of microalgae (Choricystis sp. NPA14 and Chlamydomonas sp. BM3). The average DNA yield obtained using this lysis method was between 62.5 and 78.9ng/mg for M. griffithii NS16, 42.2-247.0ng/mg for Scenedesmus sp. NS6, 70.2-110.9ng/mg for Scenedesmus sp. DPBC1 and 142.8-164.8ng/mg for Acutodesmus sp. DPBB10. DNA obtained using this method was sufficiently pure for PCR amplification. Extraction of total RNA from M. griffithii NS16 and Mychonastes sp. NPD7 using this lysis method yielded high-quality RNA suitable for RT-PCR. This lysis method is simple, cheap and would enable rapid nucleic acid extraction from freshwater microalgae without requiring costly materials and equipment such as liquid nitrogen or beadbeaters, and would facilitate molecular studies on microalgae in general.

The Effect of Plant Growth Promoting Rhizobacteria (PGPR) on Germination and Seedling Growth of Sorghum bicolor L. Moench

Germination is the first important step of the plant growth in the land. The germination and growth of sorghum in marginal land can be optimized by inoculating with PGPR (inoculants), hydrogel and CMC (carriers). The aim of the research was to evaluate the response of germination and seedling growth of sorghum using PGPR inoculation with hydrogel and CMC as carriers (in vitro) and pots containing sterile sand (in vivo). The experiment used PGPR (Azospirillum lipoferum 1103D, Azotobacter chroococcum 4103D, and Bacillus sp. 3D), and hydrogels as the reserve water, as well as carboxymethyl cellulose (CMC) as the adhesive media. The results showed that the combination of PGPR-mix (Azospirillum, Azotobacter, Bacillus) with hydrogels as bio-organic fertilizer and CMC as carriers has promoted the growth of the roots, shoots and vigour index of in vitro sorghum germination, as well as increased the root length, shoot length and total dry weight of sorghum seedlings in pots containing sterile sand. The best result of in vitro experiment (root length = 8.67 cm; shoot length = 12.6 cm; and vigor index = 2127.00) was obtained by sorghum seed inoculated with single PGPR inoculant (A. lipoferum) with carrier of CMC. The root length, shoot length and total dry weight of the highest sorghum seedlings were obtained by PGPR-mix inoculants without carriers (46.5 cm, 12 cm, and 0.477 g), PGPR-mix with carrier of CMC (48.67 cm, 15.67 cm, and 0.431 g), and PGPR-mix with carrier of hydrogel (48.67 cm, 15 cm, and 0.430 g).

Effects of arbuscular mycorrhizal fungi on early development of persimmon seedlings

Abstract The present study evaluated the effects of five species of arbuscular mycorrhizal fungi (AMF) on vegetative development parameters and nutrient uptake of persimmon (Diospyros kaki L.) seedlings. The experiment was carried out in a completely randomized experimental design with six treatments involving AMF inoculation (non-inoculated; Dentiscutata heterogama, Claroideoglomus etunicatum, Rhizophagus clarus, Acaulospora scrobiculata and A. morrowiae), in sterile soil (Oxisol – Dystrophic Red Latosol) under greenhouse conditions. Persimmon seeds were germinated in sterile sand and the seedlings were transplanted to the sterile soil and received AMF inocula. After 360 days, the following vegetative parameters were analyzed: plant height, number of leaves, leaf surface area, stem diameter, shoot and root dry mass, percentage of root colonization and nutrient accumulation in shoot and root biomass. We observed different responses to the AMF species tested. Seedling height and leaf surface area were promoted by inoculation with D. heterogama and A. morrowiae, and these species also promoted a higher percentage of root colonization. Plants inoculated with D. heterogama and C. etunicatum presented a greater number of leaves, but only D. heterogama promoted significant increases in stem diameter. Shoot dry mass was improved by inoculation with D. heterogama, A. morrowiae and C. etunicatum, while the first two species and R. clarus increased root dry mass. Nutrient accumulation in shoot and root biomass was stimulated by AMF inoculation, especially by D. heterogama and A. morrowiae. Therefore, the effects on early vegetative development were more pronounced in the persimmon seedlings inoculated with D. heterogama.